Project summary The 2014-2015 Ebola virus disease (EVD) outbreak in West Africa, caused by the Zaire Ebola virus (EBOV), resulted in over 28,000 cases and 11,000 deaths. This has been a sobering reminder of the growing threat of the filoviruses for global public health. Driven by the unprecedented dimension of this outbreak, most of the efforts to develop vaccines against the different species of ebolaviruses has been focused on EBOV. However, other ebolavirus species such as Sudan virus (SUDV) and Bundibugyo virus (BDBV) have also caused sizable outbreaks in the past 20 years and the species causing future EVD outbreaks cannot be predicted. The vaccines currently in development, including adenovirus-based vaccines and VSV-ZEBOV that was successfully tested in a Phase III clinical trial in Africa, are specific to EBOV and do not provide cross protection against SUDV or BDBV. A major obstacle for elicitation of broadly neutralizing responses is the fact that most conserved regions of the ebolavirus glycoprotein (GP), including the receptor binding site (RBS), are largely concealed on the viral surface. Using a special immunogen cocktail we have recently isolated several broadly neutralizing monoclonal antibodies (bNAbs), characterized their epitopes in collaboration with Integral Molecular, and for the first time, identified a cocktail of two antibodies that simultaneously protects against EBOV, SUDV, and BDBV. This body of knowledge can now be exploited to develop a single vaccine that protects against all ebolaviruses. Our proposal is based on two key and novel observations. i) We have identified several residues in the base of the EBOV GP trimer that, when mutated, increase the exposure of broadly neutralizing epitopes on the apical face of GP, including the RBS that is otherwise largely concealed. We have also demonstrated that immunization with such mutants broadens the antibody response towards SUDV and BDBV. ii) We have demonstrated that a proteolytically remodeled form of GP representing the post-entry form of GP in the host endosomes binds to the most potent bNAbs with very high affinity, suggesting that this ?cleaved GP? (GPCL) can be a candidate pan- ebolavirus vaccine. Building upon these observations, this Phase I project is designed in three specific Aims. In Aim 1, using the information gained from our extensive alanine scanning mutagenesis studies, a variety of mutants will be generated on the backbone of VSV-EBOV GP pseudotype virus, and their ability to elicit broadly neutralizing responses will be evaluated. Aim 2 focuses on generation and functional testing of immunogens based on GPCL. Additional specific mutations identified in Aim 1 will be incorporated into GPCL immunogen and evaluated in immunogenicity studies. In Aim 3, the best candidates identified in Aims 1 and 2 will be tested in proof of concept efficacy studies in murine challenge models of EBOV and SUDV. Upon successful proof of concept, we anticipate a phase II to demonstrate efficacy of the vaccine against EBOV, SUDV, and BDBV in non-humane primate models of infection as well as initiation of IND enabling studies.